Project Summary CRISPR-Cas systems are recently discovered RNA-based adaptive immune systems that control invasions of viruses and other mobile genetic elements in prokaryotes. CRISPR-Cas systems function by capturing short invader sequences within the CRISPR locus of the host genome, producing short crRNAs from the CRISPR, and using the crRNAs to guide Cas protein-containing effector complexes to recognize and destroy the invading nucleic acids. CRISPR-Cas based immunity is mediated by numerous and diverse Cas proteins and a given organism may possess one or more of the twelve known CRISPR-Cas modules. We know very little about how the key steps in the fascinating CRISPR-Cas immune response pathways are executed for many of the systems. Using a powerful combination of genetic, structural, and biochemical approaches, we will determine the molecular basis for how various CRISPR-Cas systems capture foreign DNA sequence within CRISPR locus memory banks to effect heritable immunity against specific invaders. We will also delineate the mechanisms by which diverse crRNA-Cas protein effector complexes selectively recognize and destroy foreign nucleic acids as a means to combat the viruses and other transgressors. The studies will provide fundamental contributions to our understanding of the range of mechanisms that have evolved to protect multitudes of prokaryotes from potentially lethal viral attack. The knowledge gained by our research will contribute directly to ongoing efforts aimed at exploiting CRISPR-Cas systems as powerful research tools (e.g. for genome editing and controlled gene expression, developing novel sequence-specific antibiotics to selectively target human pathogens (bacteria and viruses) and limiting the spread of antibiotic resistance).